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Environmental Toxicology
THE DEVELOPMENTAL NEUROTOXICITY OF PBDES: EFFECT OF DE-71 ON DOPAMINE IN ZEBRAFISH LARVAE
XIANFENG WANG, LIHUA YANG, YUANYUAN WU, CHANGJIANG HUANG, QIANGWEI WANG, JIAN HAN, YONGYONG GUO, XIONGJIE SHI, and BINGSHENG ZHOU
Environ Toxicol Chem., Accepted Article • DOI: 10.1002/etc.2906
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Environmental Toxicology
Environmental Toxicology and Chemistry DOI 10.1002/etc.2906
THE DEVELOPMENTAL NEUROTOXICITY OF PBDES: EFFECT OF DE-71 ON DOPAMINE IN ZEBRAFISH LARVAE
Running title: Effect of DE-71 on dopamine in zebrafish larvae XIANFENG WANG, †‡ LIHUA YANG,† YUANYUAN WU, § CHANGJIANG HUANG,§ QIANGWEI WANG, †‡ JIAN HAN,†‡ YONGYONG GUO, † XIONGJIE SHI, † and BINGSHENG ZHOU *†
† State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China ‡ University of Chinese Academy of Sciences, Beijing, China
§ Institute of Watershed Science and Environmental Ecology, Wenzhou Medical University, Wenzhou, China
*Address correspondence to [email protected]
Additional Supporting Information may be found in the online version of this article.
This article is protected by copyright. All rights reserved Submitted 4 September 2014; Returned for Revision 27 October 2014; Accepted 21 January 2015
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Abstract: The potential neurotoxicity of polybrominated diphenyl ethers (PBDEs) is still a great concern. In the present study, we investigated whether exposure to PBDEs could affect the neurotransmitter system and cause developmental neurotoxicity in zebrafish. Two hours post fertilization (hpf) zebrafish embryos were exposed to different concentrations of the PBDE mixture DE-71 (0-100 µg/L). The larvae were harvested at 120 hpf and the impact on dopaminergic signaling was investigated. The results revealed significant reductions of whole-body dopamine (DA) content and its metabolite, dihydroxyphenylacetic acid (DOPAC) content in DE-71-exposed larvae. The transcription of genes involved in the development of dopaminergic neurons (e.g. manf, bdnf, and nr4a2b) was significantly down-regulated upon exposure to DE-71. DE-71 also resulted in a significant decrease of tyrosine hydroxylase (TH) and dopamine transporter protein levels in dopaminergic neurons. The expression level of TH in forebrain neurons was assessed by whole-mount immunofluorescence, and the results further demonstrated that the TH protein expression level was reduced in dopaminergic neurons. In addition to these molecular changes, we also observed reduced locomotor activity in DE-71-exposed larvae. Taken together, the present study demonstrates that acute exposure to PBDEs can affect dopaminergic signaling through disrupting the synthesis and transportation of dopamine in zebrafish, thereby disrupting normal neurodevelopment. In accord with its experimental findings, this study extends our knowledge of the mechanisms governing PBDE-induced developmental neurotoxicity. This article is protected by copyright. All rights reserved Keywords: PBDEs, Neurotransmitter, Dopaminergic neuron, Developing neurotoxicity, Zebrafish larvae
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INTRODUCTION During the last decade, polybrominated diphenyl ethers (PBDEs) have attracted great attention
due to their properties as persistent organic pollutants (POPs). Of particular concern is the thyroid endocrine disruption and neurotoxicity associated with PBDE exposure in animals or humans [1]. The brain is vulnerable to insults from xenobiotics during early development [2]; additionally, it is generally observed that infants have a higher burden of PBDEs compared with adults, and young animals have been shown to have a reduced ability to excrete PBDEs [3]. Hence, the potential adverse effect of PBDEs on neurodevelopment is a major concern [1,4]. In this regard, PBDE-induced neurotoxicity has been proposed to be indirectly related to the effects of PBDEs on thyroid hormone homeostasis [1]. Conversely, others have proposed direct effects of PBDEs on the developing brain, including oxidative stress [5], interference with calcium signaling [1,6], as well as other structural and functional alterations. PBDE exposure has also been associated with changes in the expression of genes and proteins
involved in synapse and axon formation, neuronal morphology, cell migration, and synaptic plasticity [1,7]. An essential mediator of signaling across synapses is neurotransmitters; these endogenous chemicals transmit signals between neurons and other cells in the nervous system. Interestingly, environmental toxicants can induce abnormalities in neurotransmitter production or function [8], and have been implicated in the failure of central nervous system function. In this regard, a limited number of reports have shown that PBDEs may also affect neurotransmitters. For example, PBDEs can inhibit vesicular uptake of dopamine (DA) [9], affect the cholinergic This article is protected by copyright. All rights reserved
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neurotransmitter system [10], and have neurotoxic effects on DA homeostasis, glutamatergic neurotransmission, and locomotor behavior [11, 12]. In this regard, dopaminergic neurons are particularly interesting because they are known to play a critical role in motor control, cognition, motivation, and reward associated behavior, as well as endocrine regulation [13, 14]. Although several previous studies have documented an effect of PBDEs on neurotransmitter
systems, the mechanisms for these effects remain largely unknown. Additionally, it has not been reported whether and how PBDEs inhibit neurotransmitter homeostasis in early neurodevelopment. Zebrafish have proven to be a useful model system for researching the effects of toxicants on developmental neurotoxicity in the vertebrate nervous system and are becoming increasingly used in behavioral studies [15]. As such, in the present study we hypothesized that the dopaminergic neurotransmitter system of zebrafish may be vulnerable to PBDE exposure and lead to developmental neurotoxicity. We tested this hypothesis in zebrafish embryos/larvae exposed to the PBDE mixture DE-71. Our results show that PBDE exposure affects the dopaminergic system in zebrafish, which contributes to the developmental neurotoxicity of PBDEs. MATERIALS AND METHODS Chemicals
DE-71 (purity > 99.9%) was obtained from Wellington Laboratory (Ontario, Canada). Dimethyl
sulfoxide (DMSO), methanesulfonate salt, DA, and dihydroxyphenylacetic acid (DOPAC) were purchased from Sigma-Aldrich (Fluka, Shanghai, China). The PBDE standards (BDE-47, 99, 100, 153, 154), and 13C12-labeled polychlorinated biphenyl (PCB) 208, 13C12-labeled BDE-139 were purchased from AccuStandard (New Haven, CT). Trizol Reagent and PrimeScript® RT reagent Kits This article is protected by copyright. All rights reserved
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were purchased from Takara (Dalian, China). SYBR Green PCR kits were purchased from GeneCopoeia (Rockville, MD). All other chemicals used in the present study were of analytical grade.
Zebrafish embryos/larvae exposure Adult zebrafish (wild type, AB strain, 5-month old) maintenance and embryo exposure was
carried out according to published protocols [16]. Briefly, ~400 normal embryos that reached the blastula stage (2 hours post fertilization, hpf) were selected and distributed into glass beakers containing 500 mL of DE-71 (0, 3, 10, 30, or 100 µg/L). This concentration range was based on our preliminary studies that showed a trend for increased malformation rates in zebrafish embryos exposed to the highest (100 g/L) concentration of DE-71. Both the control and exposure groups received 0.005% (v/v) DMSO. There were three replicates for each exposure group and control group. The embryos were exposed until 120 hpf. During the exposure period, the exposure solutions were renewed daily. After exposure, a subset of the larvae were used for assessing locomotor activity and the others were washed five times and immediately frozen in liquid nitrogen and stored at –80°C until for analysis. The hatching, malformation and growth rates were also recorded. PBDE extraction and analysis in zebrafish larvae For PBDE analysis, ~100 larvae (5 dpf; n = 3 replicates) from each group were randomly
sampled. The extraction of PBDEs from the larvae was carried out as described previously [16]. Briefly, the larvae were weighed and freeze-dried. Prior to homogenization, a 5 ng 13C12-labeled BDE-139 was added to measure sample recovery. Next, the samples were homogenized using a mixture of acetone and isooctane (1:1, v/v) and sonicated for 30 min. The extracts were then dried This article is protected by copyright. All rights reserved
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under nitrogen, made up to a volume of 500 µl with isooctane and 4 ng 13C12-labeled PCB-208 was added as an internal standard for PBDE analysis. Quantification of PBDEs was performed using a GC (Agilent 6890, Agilent Technologies, Santa Clara, CA) equipped with a mass-selective detector (Agilent 5975C) using the electron impact (EI) mode. Procedural blanks were analyzed simultaneously with every batch of five samples to check for interference or contamination from the solvent or glassware. The recovery of 13C12-labeled BDE-139 ranged from 87% to 98%. The limit of detection was defined as a signal/noise ratio of three, and on average ranged from 0.005-0.05 ng for the PBDEs and 0.06 ng for tetra-BDE. Samples with concentrations below the detection limits were recorded as zero. Neurotransmitter analysis Neurotransmitter measurement was performed as described previously [17]. Briefly, 50 larvae
from each group (n = 3 replicates) were collected and sonicated in 200 µL of 0.1 M perchloric acid. After centrifugation for 10 min at 10,000 g at 4°C, the supernatants were filtered with a 0.22 µm polyvinylidene difluoride membrane and stored at -80°C until further analysis. DA and DOPAC contents were determined using HPLC with an ESA Colochem 5600A electrochemical detector (ESA, Chelmsford, MA, USA). Reverse phase HPLC was performed with a C18 column (250 × 3mm, 5µm). The mobile phase was methyl alcohol, acetonitrile and phosphate buffer (4:7:89), where the phosphate buffer contained 60 nM NaH2PO4, 50 µM EDTA and 2 mM sodium 1-octadecanesulfonate, pH 3.8. The flow rate was 0.8 ml/min. Quantification was made by reference to calibration curves obtained from individual monoamine standards, and concentrations were expressed in ng/g wet weight (ng/g.ww). This article is protected by copyright. All rights reserved
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Protein extraction and Western blot analysis Protein extraction and western blot analysis from ~200 zebrafish larvae from each group (n = 3
replicates), was performed as previously described [18]. Briefly, protein was extracted using commercial kits (Key-GEN BioTECH, Nanjing, China) according to the manufacturer’s instructions, and the protein concentration was measured by the bicinchoninic acid (BCA) method. Approximately 50 µg of each protein sample was loaded onto a 6% or 12% SDS-PAGE gel, then electrophoretically transferred to polyvinylidene difluoride membranes (Sigma-Aldrich). The membrane was blocked for 1 h with 5% bovine serum albumin (BSA) in tris-buffered saline (10 mM Tris, 150 mM NaCl, pH 8.0 ) at 37°C and then incubated with primary antibody against the dopamine transporter (DAT; 1:2,000), tyrosine hydroxylase (TH; 1:800) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1,000) at 4°C overnight. Both rat anti-DAT primary antibody (Chemicon, Temecula, CA) and rabbit polyclonal anti-TH (Chemicon) have been verified to be reactive with zebrafish [19, 20]. The blots were washed five times for 10 min each with tris-buffered saline Tween-20 (TBST) and then incubated with goat anti-rat (1:5,000) or anti-rabbit (1:5,000) horseradish peroxidase conjugated affinipure secondary antibodies (Proteintech, Wuhan, China) at 37°C for 1 h and then visualized with enhanced chemiluminescence using a quantitative western blot imaging system (FluorChem Q; Alpha Innotech, CA). Whole-mount immunofluorescence In addition to dopaminergic neurons, other neurons and tissues, such as noradrenergic and
adrenergic neurons, the liver, kidney, heart and gills also synthesize TH [21]. However, TH has been used as a dopaminergic marker in the vertebrate forebrain while noradrenaline or adrenaline is This article is protected by copyright. All rights reserved
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present anterior to the midbrain-hindbrain boundary in zebrafish [22]. With this in mind, we confirmed our TH western blot expression data by performing whole-mount immunofluorescence assay, as previously described [23]. In brief, the larvae were fixed in 4% paraformaldehyde overnight at 4°C, then rinsed thoroughly in phosphate buffered solution (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), dehydrated in a graded methanol series and stored in methanol at –20°C. For bleaching and to block endogenous peroxidases, larvae were incubated in 3 ml 10% H2O2 in methanol overnight at room temperature, then 10 ml PBS-T (PBS containing 0.3% Tween-20) was added, mixed and incubated for a further 24 h at room temperature. The larvae were washed five times for 20 minutes each with PBS-T and blocked for 2 h with 2% BSA in PBS-T at 37°C. Larvae were then incubated in 2% BSA in PBS-T with the primary antibody against TH (1:500) for 3 days at 4°C. After a thorough wash with PBS-T, the larvae were incubated in 2% BSA in PBS-T containing Dylight 488 conjugated Goat Anti-Rabbit (Abbkine, Redlands, CA; 1:500) for 2 h at 37°C. After washing five times for 30 min each with PBS-T and then three times for 30 min each with PBS, the larvae were imaged using Laser Scanning Confocal Microscopy (NOL-LSM 710, Zeiss, Germany) with 488 nm Argon ion laser, emission was 493-598nm and further analyzed using ImageJ software. Quantification of the staining signaling of TH neurons in the zebrafish larvae was done as previously described [24]. Briefly, the periphery of each neuronal group was outlined by manually tracing the edge. The area of each enclosed region and fluorescence signaling were detected using ImageJ software. Total staining signaling was the multiplied together of the staining area and mean staining intensity. RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR) This article is protected by copyright. All rights reserved
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Total RNA was extracted from 30 homogenized zebrafish larvae (n = 3 replicates) by using
Trizol regent (Takara) following the manufacturer’s instructions. The quality and purity of isolated RNA was examined by 1% agarose–formaldehyde gel electrophoresis with ethidium bromide staining and 260/280 nm ratios. The concentration of each RNA sample was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA). The first-strand cDNA was synthesized using a PrimeScript® RT reagent Kit (Takara, Dalian) following the manufacture’s instructions. The primer sequences for mesencephalic astrocyte derived neurotrophic factor (manf), brain-derived neurotrophic factor (bdnf), and nr4a2b, were obtained by using the online Primer 3 program (http://primer3.ut.ee/; Supporting information, Table S1). The ribosomal protein L8 (rpl8) was selected as a reference, which did not vary upon DE-71 exposure (data not shown). PCR amplification was carried out using an All-in-One qPCR Mix (GeneCopoeia) and analyzed on an ABI 7300 System (PerkinElmer Applied Biosystems, Foster City, CA). The change in the mRNA levels of the relevant genes was analyzed using the 2-ΔΔCt method. Locomotor activity measurement Larval swimming behavior was monitored under continuous light (30 min) and light-dark
transition stimulation (5 min light:5 min dark:5 min light:5 min dark) to examine their swimming speed and reaction to changes in light according to previously published methods using a ZebraLab behavior monitoring station (ViewPoint Life Sciences, Montreal, Canada) [25]. In brief, 120 hpf larvae were placed into 24-well microplates (one larva per well) in water at 28°C, and were allowed to acclimate for 10 mins before monitoring the swimming speed. A total of 30 larvae from each treatment group were assessed. The data (frequency of movements, distance travel and total duration This article is protected by copyright. All rights reserved
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of movements) were collected every 30 s and further analyzed using custom Open Office Org 2.4 software (http://www.openoffice.org). Statistical analysis All data are expressed as the mean ± standard error. The normality and the homogeneity were
verified by Kolmogorov-Smirnov and Levene’s test. Differences between the control and exposure groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s test using SPSS 13.0 software (SPSS, Chicago, IL, USA). A P-value < 0.05 was considered significantly different.
RESULTS Developmental toxicity associated with DE-71 exposure Exposure to the PBDE mixture DE-71 did not affect embryo hatching rate, survival or growth
parameters (body weight and length) (Supporting information, Table S2). The overall hatching rates were > 90%, and survival rates were > 95%. No significant difference in the malformation rate was recorded in the exposure groups, compared with the control group (Table S2). Concentration of DE-71 in exposed zebrafish larvae All five PBDE congeners (BDE-47, 99, 100, 153, and 154) were detected in the DE-71-exposed
zebrafish larvae, with BDE-47 being the predominant congener, followed by BDE-99. The burden of individual PBDE congeners and the total PBDE burden showed a concentration-dependent relationship with the exposure concentration. The total content of PBDEs in each group was 27.4 ± 0.8, 102.1 ± 7.6, 326.2 ± 10.1, and 983.7 ± 22.7 ng/g.ww. in the 3, 10, 30, and 100 µg/L exposure groups, respectively (Figure 1). Concentrations of PBDEs in the control group were below the This article is protected by copyright. All rights reserved
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detection limit (< 0.05 ng/g.w.w). Exposure to DE-71 reduces larval DA and DOPAC concentration The whole body concentration of DA and DOPAC were significantly reduced in a
concentration-dependent manner. Compared to control, DA concentration was significantly decreased by 17.9% and 21.5% in the larvae exposed to 30 and 100 µg/L DE-71, respectively (Figure 2A). Similarly, DOPAC concentration was significantly decreased by 16% and 22.3% (Figure 2B) in the larvae exposed to 30 and 100 µg/L DE-71, respectively. Exposure to DE-71 is associated with decreased protein expression levels of TH and DAT The protein expression level of TH and DAT in DE-71-exposeed zebrafish was examined by
Western blot (Figure 3). There was a concentration-dependent reduction of TH (Figure 3A and B) and DAT (Figure 3C and D) in exposed-embryos. Specifically, TH was significantly reduced by 30.3% (P < 0.01) and 56.5% (P < 0.001) in the 30 and 100 µg/L exposure groups, respectively; while DAT was significantly reduced by 20.7% (P < 0.05), 27.2% (P < 0.01) and 44.2% (P < 0.001) in the 10, 30, and 100 µg/L exposure groups, respectively, compared with those of the control group. The protein expression level of TH was also examined in the forebrain of zebrafish embryos by
whole-mount immunofluorescence. The results demonstrated that several clusters of TH-positive neurons were observed in the forebrain of zebrafish larvae in the control group at 120 hpf (Figure 4A andB). These cells could be subdivided into the following groups: olfactory bulb; subpallium; ventral thalamus; posterior tuberculum and hypothalamus. Consistent with our western blotting data, larvae exposed to DE-71 had significantly suppressed expression of TH in forebrains neurons by 36.1% (P < 0.01) and 41.1% (P < 0.001) in the 30 and 100 µg/L groups, respectively (Figure 4C). This article is protected by copyright. All rights reserved
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Exposure to DE-71 is associated with decreased mRNA levels of manf, bdnf and nr4a2b The mRNA levels of genes involved in the development of dopaminergic neurons were
examined by qRT-PCR (Figure 5). Specifically, the mRNA levels of manf and bdnf were significantly downregulated in the 10, 30, and 100 µg/L groups, respectively (all P < 0.001). A significant downregulation of the nr4a2b mRNA levels was also observed, but only in the 100 µg/L group, compared with the control (P < 0.01). Locomotor activity is decreased in DE-71-exposed larvae The locomotor activity of 5 dpf zebrafish larvae was monitored during continuous light and
during light-dark transition stimulation (Figure 6). Under continuous light conditions, the swimming speed was significantly decreased by 15.9% and 24.2% (P < 0.001) in the 30 and 100 µg/L exposed groups, respectively, compared with the control group (Figure 6A). In response to light-dark stimulation, in the first light period, the average swimming speed of the larvae was significantly decreased after exposure to 30 and 100 μg/L DE-71 (P < 0.05 and P < 0.001 respectively; Figure 6B and C). During the first dark and second light period, the average swimming speed of larvae was decreased significantly after exposure to 30 and 100 μg/L DE-71 (P < 0.001; Figure 6B and C). For the second dark period, the average swimming speed of larvae was decreased significantly after exposure to 10, 30 and 100 μg/L DE-71 (P < 0.001; Figure 6B and C). DISCUSSION Although several studies have described the neurotoxicity of PBDEs in rodents and fish, the
potential impact of PBDEs on neurotransmitter signaling is not well-known. In the present study, we used zebrafish embryos/larvae to evaluate the effect of DE-71 on dopaminergic signaling. This article is protected by copyright. All rights reserved
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Importantly, although the exposure concentrations of DE-71 used in this study was higher than those measured in the environment, the total PDBE burden in exposed zebrafish larvae in the 100 g/L group (983 ng/g.w.w) was comparable with reports of the PBDE burden in fish collected from PBDE-contaminated water (1,088 ng/g.w.w) [26], suggesting that adversely impact on dopaminergic signaling may occur in wild fish species. Our analysis of PBDE congeners in DE-71-exposed zebrafish revealed that larvae accumulated
these toxic chemicals. BDE-47 was the most abundant congener detected in exposed larvae, followed by BDE-99. This observation is consistent with a previous study in zebrafish after acute exposure to DE-71 for 5 days [27]. Interestingly, BDE-99 is the most abundant PBDE congener in commercial DE-71, and it has been reported that BDE-99 is rapidly metabolized to BDE-47 in common carp (Cyprinus carpio) [28]. Given that BDE-47 was the most abundant congener measured in DE-71-exposed larvae in our study, we interpret our data to suggest that zebrafish larvae might metabolize BDE-99 into BDE-47. In contrast to the accumulation of PBDEs in DE-71-exposed larvae, the whole body
concentration of DA and DOPAC were reduced. This result is in agreement with in vitro data obtained using cultured rat striatal synaptosomes exposed to PBDE, which found decreased DA in the synaptosomes [11], and an in vivo study in mice showing that DE-71 exposure caused a significant decrease in striatal DA and DOPAC [12]. It has also been shown that other environmental contaminants such as PCBs can affect dopaminergic signaling [29]. Similarly, the dopaminergic signaling in zebrafish has also been shown to be vulnerable to psychotropic substances and drugs [14]. Taken together, this literature indicates that the dopaminergic system is sensitive to toxicant This article is protected by copyright. All rights reserved
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exposure, and may be a common target for neurotoxicity by many toxicants. Given that 120 hpf zebrafish larvae have developed almost all their TH cell population [30], studying the dopaminergic system in these fish could serve as a good model for investigating developmental neurotoxicity. With regard to the synthesis and transport of DA, we observed that PBDE exposure significantly
downregulated the protein expression levels of both TH and DAT in the neurons of zebrafish larvae. TH is the rate limiting enzyme for DA synthesis, catalyzing the transformation of L-tyrosine to L-DOPA,
a precursor of DA [19]. Hence, downregulation of TH could have adverse effects on the
synthesis of L-DOPA, and thus the concentration of DA and will affect activity of DA neuron. Our whole-mount immunofluorescence study confirmed that the reduced expression of TH was in DA neurons. However, very little attention has been paid to neurotoxic effects of environmental toxicants on DA neurons in fish. Given the functional importance of the DA neurons, more studies are needed to understand toxic effects of chemicals on DA neurons in the environment risk assessment. DAT is a membrane transport protein that terminates DA action by its re-uptake from the
synapse [31]. The downregulation of DAT observed in the present study suggests that PBDE exposure impairs homeostasis of the dopaminergic system and affects DA neurotransmission. In agreement with our data, it has previously been shown that PCBs affect DAT expression in dopaminergic neurons. For example, exposure to the PCB mixtures Aroclor 1254 and 1260 caused a reduction in striatal DAT expression and striatal DA uptake in mice [32], supporting an association between PCB exposure and abnormal DAT function. Because DAT is important in regulating DA levels and turnover, its inhibition could increase extracellular DA concentrations, alterations in This article is protected by copyright. All rights reserved
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which might cause long-lasting dysfunction in the developing brain [33]. For example, downregulation of TH and DAT has been reported upon exposure to sodium benzoate, and such changes were related to behavioral changes in developing zebrafish larvae [34]. Taken together, this supports our hypothesis that PBDE exposure can disrupt the neurodevelopment of DA neurons, impair dopaminergic signaling and may affect locomotor behavior. In order to assess neurodevelopment in our model, we examined the expression level of several
genes involved in the development of neurons. Among the genes studied, transcriptional levels of manf and bdnf were significantly downregulated after DE-71 treatment. Manf is a dopaminergic neurotrophic factor, mainly known because of its neuroprotective functions [35]. In zebrafish, manf expression is widespread during embryonic development and can protect dopaminergic neurons from neurotoxic damage [36]. Interestingly, knockdown of manf resulted in reduced DA levels in zebrafish larvae, and the expression level of TH was also reduced [36]. Thus, downregulation of manf might have contributed to the reduced concentration of DA and TH expression observed in the present study. With regard to bdnf, it plays a fundamental role in the growth, maturation, differentiation and
maintenance of neuronal populations in the central nervous system [37]. In zebrafish embryos/larvae, bdnf is critical in neuronal development, with bdnf knockdown causing severe brain atrophy, with experimental zebrafish larvae lacking most of the neurites [38]. Thus, downregulation of bdnf observed in our study might also contribute to impaired DA neurodevelopment and decreased DA content.
The third gene we analyzed was nr4a2. This gene plays an important role in the differentiation,
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maturation and the survival of DA progenitors during zebrafish early embryogenesis (0.75 hpf – 5 dpf) [39]. However, a significant alteration in the gene expression level of nr4a2 was only observed in zebrafish larvae exposed to the highest concentration of DE-71, suggesting that it is not as sensitive to PBDE exposure as manf and bdnf. Given that the dopaminergic system is implicated in the control of locomotor function [13], and
based on the effect of DE-71 exposure described above, we hypothesized that DE-71 exposure might cause an impairment of locomotor activity in zebrafish larvae. Since neurobehavior is an integrated response of many physiological and biochemical processes, alternations of locomotor behavior could also be due to downregulation of the natural function of genes and proteins, or by direct neurotoxic effects on neuronal cells. The result of these changes in the nervous system might be translated to behavioral deficits. In the present study, we observed that DE-71 caused a significant decrease in locomotor activity in exposed larvae. This observation is in agreement with the altered behavioral activity previously reported in PBDE-exposed zebrafish larvae [26, 40]. In summary, our results demonstrate that DE-71 exposure during zebrafish embryogenesis
results in decreased expression of certain genes encoding proteins involved in DA neuronal development, decreased DA synthesis and reuptake, and an associated reduction in DA concentration. These results extend our knowledge that neurotransmitters could be a potential target for PBDE-induced developmental neurotoxicity. This study also indicates that zebrafish embryos/larvae are a suitable model for determining the effects of PBDEs and other toxicants on dopaminergic signaling and neurodevelopment. Given the co-existence of many pollutants in the environment (e.g. heavy metal; POPs), future studies into the effects of combined exposure on dopaminergic signaling This article is protected by copyright. All rights reserved
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SUPPLEMENTAL DATA Tables S1–S2. (37 KB DOC). Acknowledgment—This work was supported by the National Environmental Protection Public Welfare Science and Technology Research Program of China (No. 201309047), the National Natural Science Foundation of China (No.: 21237005, 21307153), and the State key Laboratory of Freshwater Ecology and Biotechnology (No. 2011FBZ13). Disclaimer—There is no conflict of interest. Data availability—Data, associated metadata, and calculation tools are available on request from the authors ([email protected]).
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REFERENCES 1. Dingemans MM, van den Berg M, Westerink RH. 2011. Neurotoxicity of brominated flame retardants: (in)direct effects of parent and hydroxylated polybrominated diphenyl ethers on the (developing) nervous system. Environ Health Perspect 119: 900-907.
2. Daston G, Faustman E, Ginsberg G, Fenner-Crisp P, Olin S, Sonawane B, Bruckner J, Breslin W, McLaughlin TJ. 2004. A framework for assessing risks to children from exposure to environmental agents. Environ Health Perspect 112: 238-256.
3. Staskal DF, Diliberto JJ, Birnbaum LS. 2006. Disposition of BDE 47 in developing mice. Toxicol Sci 90: 309-316.
4. Eriksson P, Jakobsson E, Fredriksson A. 2001. Brominated flame retardants: a novel class of developmental neurotoxicants in our environment? Environ Health Perspect 109: 903-908.
5. Belles M, Alonso V, Linares V, Albina ML, Sirvent JJ, Domingo JL, Sanchez DJ. 2010. Behavioral effects and oxidative status in brain regions of adult rats exposed to BDE-99. Toxicol Lett 194: 1-7.
6. Yu K, He Y, Yeung LW, Lam PK., Wu RS, Zhou B. 2008. DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells in vitro. Toxicol Sci 104: 341-351.
7. Alm H, Scholz B, Fischer C, Kultima K, Viberg H, Eriksson P, Dencker L, Stigson M. 2006. Proteomic evaluation of neonatal exposure to 2,2 ,4,4 ,5-pentabromodiphenyl ether. Environ Health Perspect 114: 254-259.
This article is protected by copyright. All rights reserved
A c c e p t e d P r e p r i nt
8. Rico EP, Rosemberg DB, Seibt K, Capiotti KM, Da Silva RS, Bonan CD. 2011. Zebrafish neurotransmitter systems as potential pharmacological and toxicological targets. Neurotoxicol Teratol 33: 608-617.
9. Dreiem A, Okoniewski RJ, Brosch KO, Miller VM, Seegal RF. 2010. Polychlorinated biphenyls and polybrominated diphenyl ethers alter striatal dopamine neurochemistry in synaptosomes from developing rats in an additive manner. Toxicol Sci 118: 150-159.
10. Viberg H, Fredriksson A, Eriksson P. 2003. Neonatal exposure to polybrominated diphenyl ether (PBDE 153) disrupts spontaneous behaviour, impairs learning and memory, and decreases hippocampal cholinergic receptors in adult mice. Toxicol Appl Pharmacol 192: 95-106.
11. Dufault C, Poles G, Driscoll LL. 2005. Brief postnatal PBDE exposure alters learning and the cholinergic modulation of attention in rats. Toxicol Sci 88: 172-180.
12. Bradner JM, Suragh TA, Wilson WW, Lazo CR, Stout KA, Kim HM, Wang MZ, Walker DI, Pennell KD, Richardson JR, Miller GW, Caudle WM. 2013. Exposure to the polybrominated diphenyl ether mixture DE-71 damages the nigrostriatal dopamine system: role of dopamine handling in neurotoxicity. Exp Neurol 241: 138-147.
13. Iversen SD, Iversen LL. 2007. Dopamine: 50 years in perspective. Trends Neurosci 30: 188-193. 14. Irons TD, Kelly PE, Hunter DL, MacPhail RC, Padilla S. 2013. Acute administration of dopaminergic drugs has differential effects on locomotion in larval zebrafish. Pharmacol Biochem Behav 103: 792-813.
15. Eddins D, Cerutti D, Williams P, Linney E, Levin ED. 2010. Zebrafish provide a sensitive model of persisting neurobehavioral effects of developmental chlorpyrifos exposure: comparison with
This article is protected by copyright. All rights reserved
A c c e p t e d P r e p r i nt
nicotine and pilocarpine effects and relationship to dopamine deficits. Neurotoxicol Teratol 32: 99-108.
16. Yu L, Deng J, Shi X, Liu C, Yu K, Zhou B. 2010. Exposure to DE-71 alters thyroid hormone levels and gene transcription in the hypothalamic-pituitary-thyroid axis of zebrafish larvae. Aquat Toxicol 97: 226-233.
17. Sallinen V, Torkko V, Sundvik M, Reenila I, Khrustalyov D, Kaslin J, Panula P. 2009. MPTP and MPP+ target specific aminergic cell populations in larval zebrafish. J Neurochem 108: 719-731.
18. Chen Q, Yu LQ, Yang LH, Zhou BS. 2012. Bioconcentration and metabolism of decabromodiphenyl ether (BDE-209) result in thyroid endocrine disruption in zebrafish larvae. Aquat Toxicol 110: 141-148.
19. Yamamoto K, Ruuskanen JO, Wullimann MF, Vernier P. 2010. Two tyrosine hydroxylase genes in vertebrates New dopaminergic territories revealed in the zebrafish brain. Mol Cell Neurosci 43: 394-402.
20. Suen MFK, Chan WS, Hung KWY, Chen YF, Mo Z.X, Yung KKL. 2013. Assessments of the effects of nicotine and ketamine using tyrosine hydroxylase-green fluorescent protein transgenic zebrafish as biosensors. Biosens Bioelectron 42: 177-185.
21. Chen YC, Priyadarshini M, Panula P. 2009. Complementary developmental expression of the two tyrosine hydroxylase transcripts in zebrafish. Histochem Cell Biol 132: 375-381.
22. Tay TL, Ronneberger O, Ryu S, Nitschke R, Driever W. 2011. Comprehensive catecholaminergic projectome analysis reveals single-neuron integration of zebrafish ascending and descending dopaminergic systems. Nat Commun 2.
This article is protected by copyright. All rights reserved
A c c e p t e d P r e p r i nt
23. Elsalini OA, Rohr KB. 2003. Phenylthiourea disrupts thyroid function in developing zebrafish. Dev Genes Evol 212: 593-598.
24. Parng C, Roy NM, Ton C, Lin Y, McGrath P. 2007. Neurotoxicity assessment using zebrafish. J. Pharmacol. Toxicol. Methods 55: 103-112.
25. He J, Yang D, Wang C, Liu W, Liao J, Xu T, Bai C, Chen J, Lin K, Huang C, Dong Q. 2011. Chronic zebrafish low dose decabrominated diphenyl ether (BDE-209) exposure affected parental gonad development and locomotion in F1 offspring. Ecotoxicology 20: 1813-1822.
26. Luo Q, Cai ZW, Wong MH. 2007. Polybrominated diphenyl ethers in fish and sediment from river polluted by electronic waste. Sci Total Environ 383: 115-127.
27. Chen L, Huang C, Hu C, Yu K, Yang L, Zhou B. 2012. Acute exposure to DE-71: Effects on locomotor behavior and developmental neurotoxicity in zebrafish larvae. Environ. Toxicol Chem 31: 2338-2344.
28. Stapleton HM, Letcher RJ, Baker JE. 2004. Debromination of polybrominated diphenyl ether congeners BDE 99 and BDE 183 in the intestinal tract of the common carp (Cyprinus carpio). Environ Sci Technol 38: 1054-1061.
29. Mariussen E, Fonnum F. 2001. The effect of polychlorinated biphenyls on the high affinity uptake of the neurotransmitters, dopamine, serotonin, glutamate and GABA, into rat brain synaptosomes. Toxicology 159: 11-21.
30. Rink E, Wullimann MF. 2002. Development of the catecholaminergic system in the early zebrafish brain: an immunohistochemical study. Dev Brain Res 137: 89-100.
31. Jaber M, Jones S, Giros B, Caron MG. 1997. The dopamine transporter: A crucial component This article is protected by copyright. All rights reserved
A c c e p t e d P r e p r i nt
regulating dopamine transmission. Movement Disord 12: 629-633.
32. Caudle WM, Richardson JR, Delea KC, Guillot TS, Wang MZ, Pennell KD, Miller GW. 2006. Polychlorinated biphenyl-induced reduction of dopamine transporter expression as a precursor to Parkinson's disease-associated dopamine toxicity. Toxicol Sci 92: 490-499.
33. Buske C, Gerlai R. 2011. Early embryonic ethanol exposure impairs shoaling and the dopaminergic and serotoninergic systems in adult zebrafish. Neurotoxicol Teratol 33: 698-707.
34. Chen Q, Huang N, Huang J, Chen S, Fan J, Li C, Xie F. 2009. Sodium benzoate exposure downregulates the expression of tyrosine hydroxylase and dopamine transporter in dopaminergic neurons in developing zebrafish. Birth Defects Res Part B, Dev Reprod Toxicol 86: 85-91.
35. Airavaara M, Shen H, Kuo CC, Peranen J, Saarma M, Hoffer B, Wang Y. 2009. Mesencephalic Astrocyte-Derived Neurotrophic Factor Reduces Ischemic Brain Injury and Promotes Behavioral Recovery in Rats. J Comp Neurol 515: 116-124.
36. Chen Y, Sundvik M, Rozov S, Priyadarshini M, Panula P. 2012. MANF regulates dopaminergic neuron development in larval zebrafish. Dev Biol 370: 237-249.
37. De Felice E, Porreca I, Alleva E, De Girolamo P, Ambrosino C, Ciriaco E, Germana A, Sordino P. 2014. Localization of BDNF expression in the developing brain of zebrafish. J Anat 224: 564-574.
38. Diekmann H, Anichtchik O, Fleming A, Futter M, Goldsmith P, Roach A, Rubinsztein DC. 2009. Decreased BDNF levels are a major contributor to the embryonic phenotype of huntingtin knockdown zebrafish. J Neurosci 29: 1343-1349.
This article is protected by copyright. All rights reserved
A c c e p t e d P r e p r i nt
39. Luo G, Chen Y, Li X, Liu T, Le W. 2008. Nr4a2 is essential for the differentiation of dopaminergic neurons during zebrafish embryogenesis. Mol Cell Neurosci 39: 202-210.
40. Usenko CY, Robinson EM, Usenko S, Brooks BW, Bruce ED. 2011. PBDE developmental effects on embryonic zebrafish. Environ Toxicol Chem 30: 1865-1872.
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Figure 1. PBDEs accumulate in zebrafish larvae exposed to DE-71. The concentration of different PBDE congeners were measured in zebrafish larvae exposed to 0, 3, 10, 30, and 100 g/L DE-71 for 5 days. n = 3 replicate samples; each sample contained 100 larvae; data expressed as mean ± SEM. Figure 2. DE-71 exposure is associated with a reduced whole-body content of dopamine and dihydroxyphenylacetic
acid.
The
concentration
of
(A)
dopamine
(DA)
and
(B)
dihydroxyphenylacetic acid (DOPAC) were measured in zebrafish larvae exposed to 0, 3, 10, 30, and 100 g/L DE-71 for 5 days. n = 3 replicate samples; each sample contained 50 larvae; data expressed as mean ± SEM; *P < 0.05, and ** P < 0.01 indicate significant differences between the exposed larvae and the control group. Figure 3. DE-71 exposure is associated with reduced protein expression levels of tyrosine hydroxylase and the dopamine transporter. Western blot analysis was used to measure the protein expression level of tyrosine hydroxylase (TH; A and B) and the dopamine transporter (DAT; C and D) in zebrafish larvae exposed to 0, 3, 10, 30, and 100 g/L of DE-71 for 5 days. Upper panel shows a representative western blot and the lower panel shows quantification of three replicate samples, normalized to GAPDH expression; data expressed as mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001 indicate significant differences from the control group. Figure 4. Decreased tyrosine hydroxylase expression in the forebrain of zebrafish larvae exposed to DE-71. Whole-mount immunofluorescence was used to assess tyrosine hydroxylase (TH) expression of dopaminergic neurons in zebrafish larvae after exposure to 0, 3, 10, 30, and 100 g/L of DE-71 for 5 days. (A) The control larvae; (B) 100 g/L DE-71 exposure larvae; (C) TH expression levels are indicated by fold change of fluorescence intensity relative to the control. The abbreviations are used: olfactory bulb (Ob), telencephalon (Tel), ventral diencephalon (vDc) and caudal hypothalamus (Hc). Data are presented as mean ± SEM from 6 individual larvae in each group; ** P < 0.01; *** P < 0.001 significant difference from the control group. Figure 5. Altered expression of neurodevelopmental genes in zebrafish larvae exposed to DE-71. The mRNA expression level of manf, bdnf and nr4a2b were significantly decreased following exposure 0,
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3, 10, 30, and 100 g/L DE-71 for 5 days in a concentration dependent manner. Data presented as mean ± SEM; n = 3 replicate samples; each sample contained 30 larvae; ** P < 0.01, *** P < 0.001 indicate significant differences between the exposed larvae and the control group. Figure 6. Abnormal locomotor behavior in zebrafish larvae exposed to DE-71. Locomotor behavior was assessed in zebrafish larvae exposed to 0, 3, 10, 30, or 100 µg/L DE-71 for 5 days. The following parameters were measured: average swimming speed during a continuous light test (A); locomotor traces of the larvae (B); and average swimming speed of the larvae during a light-dark-light-dark photoperiod stimulation test (C). Data are expressed as the mean ± SEM of three replicates (ten larvae per replicate) in 30-s intervals; * P < 0.05, *** P < 0.001 indicate significant difference between the exposed larvae and the control group.
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Environmental Toxicology
THE DEVELOPMENTAL NEUROTOXICITY OF PBDES: EFFECT OF DE-71 ON DOPAMINE IN ZEBRAFISH LARVAE
XIANFENG WANG, LIHUA YANG, YUANYUAN WU, CHANGJIANG HUANG, QIANGWEI WANG, JIAN HAN, YONGYONG GUO, XIONGJIE SHI, and BINGSHENG ZHOU
Environ Toxicol Chem., Accepted Article • DOI: 10.1002/etc.2906
Accepted Article
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Environmental Toxicology
Environmental Toxicology and Chemistry DOI 10.1002/etc.2906
THE DEVELOPMENTAL NEUROTOXICITY OF PBDES: EFFECT OF DE-71 ON DOPAMINE IN ZEBRAFISH LARVAE
Running title: Effect of DE-71 on dopamine in zebrafish larvae XIANFENG WANG, †‡ LIHUA YANG,† YUANYUAN WU, § CHANGJIANG HUANG,§ QIANGWEI WANG, †‡ JIAN HAN,†‡ YONGYONG GUO, † XIONGJIE SHI, † and BINGSHENG ZHOU *†
† State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China ‡ University of Chinese Academy of Sciences, Beijing, China
§ Institute of Watershed Science and Environmental Ecology, Wenzhou Medical University, Wenzhou, China
*Address correspondence to [email protected]
Additional Supporting Information may be found in the online version of this article.
This article is protected by copyright. All rights reserved Submitted 4 September 2014; Returned for Revision 27 October 2014; Accepted 21 January 2015
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Abstract: The potential neurotoxicity of polybrominated diphenyl ethers (PBDEs) is still a great concern. In the present study, we investigated whether exposure to PBDEs could affect the neurotransmitter system and cause developmental neurotoxicity in zebrafish. Two hours post fertilization (hpf) zebrafish embryos were exposed to different concentrations of the PBDE mixture DE-71 (0-100 µg/L). The larvae were harvested at 120 hpf and the impact on dopaminergic signaling was investigated. The results revealed significant reductions of whole-body dopamine (DA) content and its metabolite, dihydroxyphenylacetic acid (DOPAC) content in DE-71-exposed larvae. The transcription of genes involved in the development of dopaminergic neurons (e.g. manf, bdnf, and nr4a2b) was significantly down-regulated upon exposure to DE-71. DE-71 also resulted in a significant decrease of tyrosine hydroxylase (TH) and dopamine transporter protein levels in dopaminergic neurons. The expression level of TH in forebrain neurons was assessed by whole-mount immunofluorescence, and the results further demonstrated that the TH protein expression level was reduced in dopaminergic neurons. In addition to these molecular changes, we also observed reduced locomotor activity in DE-71-exposed larvae. Taken together, the present study demonstrates that acute exposure to PBDEs can affect dopaminergic signaling through disrupting the synthesis and transportation of dopamine in zebrafish, thereby disrupting normal neurodevelopment. In accord with its experimental findings, this study extends our knowledge of the mechanisms governing PBDE-induced developmental neurotoxicity. This article is protected by copyright. All rights reserved Keywords: PBDEs, Neurotransmitter, Dopaminergic neuron, Developing neurotoxicity, Zebrafish larvae
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INTRODUCTION During the last decade, polybrominated diphenyl ethers (PBDEs) have attracted great attention
due to their properties as persistent organic pollutants (POPs). Of particular concern is the thyroid endocrine disruption and neurotoxicity associated with PBDE exposure in animals or humans [1]. The brain is vulnerable to insults from xenobiotics during early development [2]; additionally, it is generally observed that infants have a higher burden of PBDEs compared with adults, and young animals have been shown to have a reduced ability to excrete PBDEs [3]. Hence, the potential adverse effect of PBDEs on neurodevelopment is a major concern [1,4]. In this regard, PBDE-induced neurotoxicity has been proposed to be indirectly related to the effects of PBDEs on thyroid hormone homeostasis [1]. Conversely, others have proposed direct effects of PBDEs on the developing brain, including oxidative stress [5], interference with calcium signaling [1,6], as well as other structural and functional alterations. PBDE exposure has also been associated with changes in the expression of genes and proteins
involved in synapse and axon formation, neuronal morphology, cell migration, and synaptic plasticity [1,7]. An essential mediator of signaling across synapses is neurotransmitters; these endogenous chemicals transmit signals between neurons and other cells in the nervous system. Interestingly, environmental toxicants can induce abnormalities in neurotransmitter production or function [8], and have been implicated in the failure of central nervous system function. In this regard, a limited number of reports have shown that PBDEs may also affect neurotransmitters. For example, PBDEs can inhibit vesicular uptake of dopamine (DA) [9], affect the cholinergic This article is protected by copyright. All rights reserved
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neurotransmitter system [10], and have neurotoxic effects on DA homeostasis, glutamatergic neurotransmission, and locomotor behavior [11, 12]. In this regard, dopaminergic neurons are particularly interesting because they are known to play a critical role in motor control, cognition, motivation, and reward associated behavior, as well as endocrine regulation [13, 14]. Although several previous studies have documented an effect of PBDEs on neurotransmitter
systems, the mechanisms for these effects remain largely unknown. Additionally, it has not been reported whether and how PBDEs inhibit neurotransmitter homeostasis in early neurodevelopment. Zebrafish have proven to be a useful model system for researching the effects of toxicants on developmental neurotoxicity in the vertebrate nervous system and are becoming increasingly used in behavioral studies [15]. As such, in the present study we hypothesized that the dopaminergic neurotransmitter system of zebrafish may be vulnerable to PBDE exposure and lead to developmental neurotoxicity. We tested this hypothesis in zebrafish embryos/larvae exposed to the PBDE mixture DE-71. Our results show that PBDE exposure affects the dopaminergic system in zebrafish, which contributes to the developmental neurotoxicity of PBDEs. MATERIALS AND METHODS Chemicals
DE-71 (purity > 99.9%) was obtained from Wellington Laboratory (Ontario, Canada). Dimethyl
sulfoxide (DMSO), methanesulfonate salt, DA, and dihydroxyphenylacetic acid (DOPAC) were purchased from Sigma-Aldrich (Fluka, Shanghai, China). The PBDE standards (BDE-47, 99, 100, 153, 154), and 13C12-labeled polychlorinated biphenyl (PCB) 208, 13C12-labeled BDE-139 were purchased from AccuStandard (New Haven, CT). Trizol Reagent and PrimeScript® RT reagent Kits This article is protected by copyright. All rights reserved
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were purchased from Takara (Dalian, China). SYBR Green PCR kits were purchased from GeneCopoeia (Rockville, MD). All other chemicals used in the present study were of analytical grade.
Zebrafish embryos/larvae exposure Adult zebrafish (wild type, AB strain, 5-month old) maintenance and embryo exposure was
carried out according to published protocols [16]. Briefly, ~400 normal embryos that reached the blastula stage (2 hours post fertilization, hpf) were selected and distributed into glass beakers containing 500 mL of DE-71 (0, 3, 10, 30, or 100 µg/L). This concentration range was based on our preliminary studies that showed a trend for increased malformation rates in zebrafish embryos exposed to the highest (100 g/L) concentration of DE-71. Both the control and exposure groups received 0.005% (v/v) DMSO. There were three replicates for each exposure group and control group. The embryos were exposed until 120 hpf. During the exposure period, the exposure solutions were renewed daily. After exposure, a subset of the larvae were used for assessing locomotor activity and the others were washed five times and immediately frozen in liquid nitrogen and stored at –80°C until for analysis. The hatching, malformation and growth rates were also recorded. PBDE extraction and analysis in zebrafish larvae For PBDE analysis, ~100 larvae (5 dpf; n = 3 replicates) from each group were randomly
sampled. The extraction of PBDEs from the larvae was carried out as described previously [16]. Briefly, the larvae were weighed and freeze-dried. Prior to homogenization, a 5 ng 13C12-labeled BDE-139 was added to measure sample recovery. Next, the samples were homogenized using a mixture of acetone and isooctane (1:1, v/v) and sonicated for 30 min. The extracts were then dried This article is protected by copyright. All rights reserved
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under nitrogen, made up to a volume of 500 µl with isooctane and 4 ng 13C12-labeled PCB-208 was added as an internal standard for PBDE analysis. Quantification of PBDEs was performed using a GC (Agilent 6890, Agilent Technologies, Santa Clara, CA) equipped with a mass-selective detector (Agilent 5975C) using the electron impact (EI) mode. Procedural blanks were analyzed simultaneously with every batch of five samples to check for interference or contamination from the solvent or glassware. The recovery of 13C12-labeled BDE-139 ranged from 87% to 98%. The limit of detection was defined as a signal/noise ratio of three, and on average ranged from 0.005-0.05 ng for the PBDEs and 0.06 ng for tetra-BDE. Samples with concentrations below the detection limits were recorded as zero. Neurotransmitter analysis Neurotransmitter measurement was performed as described previously [17]. Briefly, 50 larvae
from each group (n = 3 replicates) were collected and sonicated in 200 µL of 0.1 M perchloric acid. After centrifugation for 10 min at 10,000 g at 4°C, the supernatants were filtered with a 0.22 µm polyvinylidene difluoride membrane and stored at -80°C until further analysis. DA and DOPAC contents were determined using HPLC with an ESA Colochem 5600A electrochemical detector (ESA, Chelmsford, MA, USA). Reverse phase HPLC was performed with a C18 column (250 × 3mm, 5µm). The mobile phase was methyl alcohol, acetonitrile and phosphate buffer (4:7:89), where the phosphate buffer contained 60 nM NaH2PO4, 50 µM EDTA and 2 mM sodium 1-octadecanesulfonate, pH 3.8. The flow rate was 0.8 ml/min. Quantification was made by reference to calibration curves obtained from individual monoamine standards, and concentrations were expressed in ng/g wet weight (ng/g.ww). This article is protected by copyright. All rights reserved
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Protein extraction and Western blot analysis Protein extraction and western blot analysis from ~200 zebrafish larvae from each group (n = 3
replicates), was performed as previously described [18]. Briefly, protein was extracted using commercial kits (Key-GEN BioTECH, Nanjing, China) according to the manufacturer’s instructions, and the protein concentration was measured by the bicinchoninic acid (BCA) method. Approximately 50 µg of each protein sample was loaded onto a 6% or 12% SDS-PAGE gel, then electrophoretically transferred to polyvinylidene difluoride membranes (Sigma-Aldrich). The membrane was blocked for 1 h with 5% bovine serum albumin (BSA) in tris-buffered saline (10 mM Tris, 150 mM NaCl, pH 8.0 ) at 37°C and then incubated with primary antibody against the dopamine transporter (DAT; 1:2,000), tyrosine hydroxylase (TH; 1:800) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1,000) at 4°C overnight. Both rat anti-DAT primary antibody (Chemicon, Temecula, CA) and rabbit polyclonal anti-TH (Chemicon) have been verified to be reactive with zebrafish [19, 20]. The blots were washed five times for 10 min each with tris-buffered saline Tween-20 (TBST) and then incubated with goat anti-rat (1:5,000) or anti-rabbit (1:5,000) horseradish peroxidase conjugated affinipure secondary antibodies (Proteintech, Wuhan, China) at 37°C for 1 h and then visualized with enhanced chemiluminescence using a quantitative western blot imaging system (FluorChem Q; Alpha Innotech, CA). Whole-mount immunofluorescence In addition to dopaminergic neurons, other neurons and tissues, such as noradrenergic and
adrenergic neurons, the liver, kidney, heart and gills also synthesize TH [21]. However, TH has been used as a dopaminergic marker in the vertebrate forebrain while noradrenaline or adrenaline is This article is protected by copyright. All rights reserved
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present anterior to the midbrain-hindbrain boundary in zebrafish [22]. With this in mind, we confirmed our TH western blot expression data by performing whole-mount immunofluorescence assay, as previously described [23]. In brief, the larvae were fixed in 4% paraformaldehyde overnight at 4°C, then rinsed thoroughly in phosphate buffered solution (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), dehydrated in a graded methanol series and stored in methanol at –20°C. For bleaching and to block endogenous peroxidases, larvae were incubated in 3 ml 10% H2O2 in methanol overnight at room temperature, then 10 ml PBS-T (PBS containing 0.3% Tween-20) was added, mixed and incubated for a further 24 h at room temperature. The larvae were washed five times for 20 minutes each with PBS-T and blocked for 2 h with 2% BSA in PBS-T at 37°C. Larvae were then incubated in 2% BSA in PBS-T with the primary antibody against TH (1:500) for 3 days at 4°C. After a thorough wash with PBS-T, the larvae were incubated in 2% BSA in PBS-T containing Dylight 488 conjugated Goat Anti-Rabbit (Abbkine, Redlands, CA; 1:500) for 2 h at 37°C. After washing five times for 30 min each with PBS-T and then three times for 30 min each with PBS, the larvae were imaged using Laser Scanning Confocal Microscopy (NOL-LSM 710, Zeiss, Germany) with 488 nm Argon ion laser, emission was 493-598nm and further analyzed using ImageJ software. Quantification of the staining signaling of TH neurons in the zebrafish larvae was done as previously described [24]. Briefly, the periphery of each neuronal group was outlined by manually tracing the edge. The area of each enclosed region and fluorescence signaling were detected using ImageJ software. Total staining signaling was the multiplied together of the staining area and mean staining intensity. RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR) This article is protected by copyright. All rights reserved
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Total RNA was extracted from 30 homogenized zebrafish larvae (n = 3 replicates) by using
Trizol regent (Takara) following the manufacturer’s instructions. The quality and purity of isolated RNA was examined by 1% agarose–formaldehyde gel electrophoresis with ethidium bromide staining and 260/280 nm ratios. The concentration of each RNA sample was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA). The first-strand cDNA was synthesized using a PrimeScript® RT reagent Kit (Takara, Dalian) following the manufacture’s instructions. The primer sequences for mesencephalic astrocyte derived neurotrophic factor (manf), brain-derived neurotrophic factor (bdnf), and nr4a2b, were obtained by using the online Primer 3 program (http://primer3.ut.ee/; Supporting information, Table S1). The ribosomal protein L8 (rpl8) was selected as a reference, which did not vary upon DE-71 exposure (data not shown). PCR amplification was carried out using an All-in-One qPCR Mix (GeneCopoeia) and analyzed on an ABI 7300 System (PerkinElmer Applied Biosystems, Foster City, CA). The change in the mRNA levels of the relevant genes was analyzed using the 2-ΔΔCt method. Locomotor activity measurement Larval swimming behavior was monitored under continuous light (30 min) and light-dark
transition stimulation (5 min light:5 min dark:5 min light:5 min dark) to examine their swimming speed and reaction to changes in light according to previously published methods using a ZebraLab behavior monitoring station (ViewPoint Life Sciences, Montreal, Canada) [25]. In brief, 120 hpf larvae were placed into 24-well microplates (one larva per well) in water at 28°C, and were allowed to acclimate for 10 mins before monitoring the swimming speed. A total of 30 larvae from each treatment group were assessed. The data (frequency of movements, distance travel and total duration This article is protected by copyright. All rights reserved
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of movements) were collected every 30 s and further analyzed using custom Open Office Org 2.4 software (http://www.openoffice.org). Statistical analysis All data are expressed as the mean ± standard error. The normality and the homogeneity were
verified by Kolmogorov-Smirnov and Levene’s test. Differences between the control and exposure groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s test using SPSS 13.0 software (SPSS, Chicago, IL, USA). A P-value < 0.05 was considered significantly different.
RESULTS Developmental toxicity associated with DE-71 exposure Exposure to the PBDE mixture DE-71 did not affect embryo hatching rate, survival or growth
parameters (body weight and length) (Supporting information, Table S2). The overall hatching rates were > 90%, and survival rates were > 95%. No significant difference in the malformation rate was recorded in the exposure groups, compared with the control group (Table S2). Concentration of DE-71 in exposed zebrafish larvae All five PBDE congeners (BDE-47, 99, 100, 153, and 154) were detected in the DE-71-exposed
zebrafish larvae, with BDE-47 being the predominant congener, followed by BDE-99. The burden of individual PBDE congeners and the total PBDE burden showed a concentration-dependent relationship with the exposure concentration. The total content of PBDEs in each group was 27.4 ± 0.8, 102.1 ± 7.6, 326.2 ± 10.1, and 983.7 ± 22.7 ng/g.ww. in the 3, 10, 30, and 100 µg/L exposure groups, respectively (Figure 1). Concentrations of PBDEs in the control group were below the This article is protected by copyright. All rights reserved
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detection limit (< 0.05 ng/g.w.w). Exposure to DE-71 reduces larval DA and DOPAC concentration The whole body concentration of DA and DOPAC were significantly reduced in a
concentration-dependent manner. Compared to control, DA concentration was significantly decreased by 17.9% and 21.5% in the larvae exposed to 30 and 100 µg/L DE-71, respectively (Figure 2A). Similarly, DOPAC concentration was significantly decreased by 16% and 22.3% (Figure 2B) in the larvae exposed to 30 and 100 µg/L DE-71, respectively. Exposure to DE-71 is associated with decreased protein expression levels of TH and DAT The protein expression level of TH and DAT in DE-71-exposeed zebrafish was examined by
Western blot (Figure 3). There was a concentration-dependent reduction of TH (Figure 3A and B) and DAT (Figure 3C and D) in exposed-embryos. Specifically, TH was significantly reduced by 30.3% (P < 0.01) and 56.5% (P < 0.001) in the 30 and 100 µg/L exposure groups, respectively; while DAT was significantly reduced by 20.7% (P < 0.05), 27.2% (P < 0.01) and 44.2% (P < 0.001) in the 10, 30, and 100 µg/L exposure groups, respectively, compared with those of the control group. The protein expression level of TH was also examined in the forebrain of zebrafish embryos by
whole-mount immunofluorescence. The results demonstrated that several clusters of TH-positive neurons were observed in the forebrain of zebrafish larvae in the control group at 120 hpf (Figure 4A andB). These cells could be subdivided into the following groups: olfactory bulb; subpallium; ventral thalamus; posterior tuberculum and hypothalamus. Consistent with our western blotting data, larvae exposed to DE-71 had significantly suppressed expression of TH in forebrains neurons by 36.1% (P < 0.01) and 41.1% (P < 0.001) in the 30 and 100 µg/L groups, respectively (Figure 4C). This article is protected by copyright. All rights reserved
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Exposure to DE-71 is associated with decreased mRNA levels of manf, bdnf and nr4a2b The mRNA levels of genes involved in the development of dopaminergic neurons were
examined by qRT-PCR (Figure 5). Specifically, the mRNA levels of manf and bdnf were significantly downregulated in the 10, 30, and 100 µg/L groups, respectively (all P < 0.001). A significant downregulation of the nr4a2b mRNA levels was also observed, but only in the 100 µg/L group, compared with the control (P < 0.01). Locomotor activity is decreased in DE-71-exposed larvae The locomotor activity of 5 dpf zebrafish larvae was monitored during continuous light and
during light-dark transition stimulation (Figure 6). Under continuous light conditions, the swimming speed was significantly decreased by 15.9% and 24.2% (P < 0.001) in the 30 and 100 µg/L exposed groups, respectively, compared with the control group (Figure 6A). In response to light-dark stimulation, in the first light period, the average swimming speed of the larvae was significantly decreased after exposure to 30 and 100 μg/L DE-71 (P < 0.05 and P < 0.001 respectively; Figure 6B and C). During the first dark and second light period, the average swimming speed of larvae was decreased significantly after exposure to 30 and 100 μg/L DE-71 (P < 0.001; Figure 6B and C). For the second dark period, the average swimming speed of larvae was decreased significantly after exposure to 10, 30 and 100 μg/L DE-71 (P < 0.001; Figure 6B and C). DISCUSSION Although several studies have described the neurotoxicity of PBDEs in rodents and fish, the
potential impact of PBDEs on neurotransmitter signaling is not well-known. In the present study, we used zebrafish embryos/larvae to evaluate the effect of DE-71 on dopaminergic signaling. This article is protected by copyright. All rights reserved
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Importantly, although the exposure concentrations of DE-71 used in this study was higher than those measured in the environment, the total PDBE burden in exposed zebrafish larvae in the 100 g/L group (983 ng/g.w.w) was comparable with reports of the PBDE burden in fish collected from PBDE-contaminated water (1,088 ng/g.w.w) [26], suggesting that adversely impact on dopaminergic signaling may occur in wild fish species. Our analysis of PBDE congeners in DE-71-exposed zebrafish revealed that larvae accumulated
these toxic chemicals. BDE-47 was the most abundant congener detected in exposed larvae, followed by BDE-99. This observation is consistent with a previous study in zebrafish after acute exposure to DE-71 for 5 days [27]. Interestingly, BDE-99 is the most abundant PBDE congener in commercial DE-71, and it has been reported that BDE-99 is rapidly metabolized to BDE-47 in common carp (Cyprinus carpio) [28]. Given that BDE-47 was the most abundant congener measured in DE-71-exposed larvae in our study, we interpret our data to suggest that zebrafish larvae might metabolize BDE-99 into BDE-47. In contrast to the accumulation of PBDEs in DE-71-exposed larvae, the whole body
concentration of DA and DOPAC were reduced. This result is in agreement with in vitro data obtained using cultured rat striatal synaptosomes exposed to PBDE, which found decreased DA in the synaptosomes [11], and an in vivo study in mice showing that DE-71 exposure caused a significant decrease in striatal DA and DOPAC [12]. It has also been shown that other environmental contaminants such as PCBs can affect dopaminergic signaling [29]. Similarly, the dopaminergic signaling in zebrafish has also been shown to be vulnerable to psychotropic substances and drugs [14]. Taken together, this literature indicates that the dopaminergic system is sensitive to toxicant This article is protected by copyright. All rights reserved
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exposure, and may be a common target for neurotoxicity by many toxicants. Given that 120 hpf zebrafish larvae have developed almost all their TH cell population [30], studying the dopaminergic system in these fish could serve as a good model for investigating developmental neurotoxicity. With regard to the synthesis and transport of DA, we observed that PBDE exposure significantly
downregulated the protein expression levels of both TH and DAT in the neurons of zebrafish larvae. TH is the rate limiting enzyme for DA synthesis, catalyzing the transformation of L-tyrosine to L-DOPA,
a precursor of DA [19]. Hence, downregulation of TH could have adverse effects on the
synthesis of L-DOPA, and thus the concentration of DA and will affect activity of DA neuron. Our whole-mount immunofluorescence study confirmed that the reduced expression of TH was in DA neurons. However, very little attention has been paid to neurotoxic effects of environmental toxicants on DA neurons in fish. Given the functional importance of the DA neurons, more studies are needed to understand toxic effects of chemicals on DA neurons in the environment risk assessment. DAT is a membrane transport protein that terminates DA action by its re-uptake from the
synapse [31]. The downregulation of DAT observed in the present study suggests that PBDE exposure impairs homeostasis of the dopaminergic system and affects DA neurotransmission. In agreement with our data, it has previously been shown that PCBs affect DAT expression in dopaminergic neurons. For example, exposure to the PCB mixtures Aroclor 1254 and 1260 caused a reduction in striatal DAT expression and striatal DA uptake in mice [32], supporting an association between PCB exposure and abnormal DAT function. Because DAT is important in regulating DA levels and turnover, its inhibition could increase extracellular DA concentrations, alterations in This article is protected by copyright. All rights reserved
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which might cause long-lasting dysfunction in the developing brain [33]. For example, downregulation of TH and DAT has been reported upon exposure to sodium benzoate, and such changes were related to behavioral changes in developing zebrafish larvae [34]. Taken together, this supports our hypothesis that PBDE exposure can disrupt the neurodevelopment of DA neurons, impair dopaminergic signaling and may affect locomotor behavior. In order to assess neurodevelopment in our model, we examined the expression level of several
genes involved in the development of neurons. Among the genes studied, transcriptional levels of manf and bdnf were significantly downregulated after DE-71 treatment. Manf is a dopaminergic neurotrophic factor, mainly known because of its neuroprotective functions [35]. In zebrafish, manf expression is widespread during embryonic development and can protect dopaminergic neurons from neurotoxic damage [36]. Interestingly, knockdown of manf resulted in reduced DA levels in zebrafish larvae, and the expression level of TH was also reduced [36]. Thus, downregulation of manf might have contributed to the reduced concentration of DA and TH expression observed in the present study. With regard to bdnf, it plays a fundamental role in the growth, maturation, differentiation and
maintenance of neuronal populations in the central nervous system [37]. In zebrafish embryos/larvae, bdnf is critical in neuronal development, with bdnf knockdown causing severe brain atrophy, with experimental zebrafish larvae lacking most of the neurites [38]. Thus, downregulation of bdnf observed in our study might also contribute to impaired DA neurodevelopment and decreased DA content.
The third gene we analyzed was nr4a2. This gene plays an important role in the differentiation,
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maturation and the survival of DA progenitors during zebrafish early embryogenesis (0.75 hpf – 5 dpf) [39]. However, a significant alteration in the gene expression level of nr4a2 was only observed in zebrafish larvae exposed to the highest concentration of DE-71, suggesting that it is not as sensitive to PBDE exposure as manf and bdnf. Given that the dopaminergic system is implicated in the control of locomotor function [13], and
based on the effect of DE-71 exposure described above, we hypothesized that DE-71 exposure might cause an impairment of locomotor activity in zebrafish larvae. Since neurobehavior is an integrated response of many physiological and biochemical processes, alternations of locomotor behavior could also be due to downregulation of the natural function of genes and proteins, or by direct neurotoxic effects on neuronal cells. The result of these changes in the nervous system might be translated to behavioral deficits. In the present study, we observed that DE-71 caused a significant decrease in locomotor activity in exposed larvae. This observation is in agreement with the altered behavioral activity previously reported in PBDE-exposed zebrafish larvae [26, 40]. In summary, our results demonstrate that DE-71 exposure during zebrafish embryogenesis
results in decreased expression of certain genes encoding proteins involved in DA neuronal development, decreased DA synthesis and reuptake, and an associated reduction in DA concentration. These results extend our knowledge that neurotransmitters could be a potential target for PBDE-induced developmental neurotoxicity. This study also indicates that zebrafish embryos/larvae are a suitable model for determining the effects of PBDEs and other toxicants on dopaminergic signaling and neurodevelopment. Given the co-existence of many pollutants in the environment (e.g. heavy metal; POPs), future studies into the effects of combined exposure on dopaminergic signaling This article is protected by copyright. All rights reserved
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SUPPLEMENTAL DATA Tables S1–S2. (37 KB DOC). Acknowledgment—This work was supported by the National Environmental Protection Public Welfare Science and Technology Research Program of China (No. 201309047), the National Natural Science Foundation of China (No.: 21237005, 21307153), and the State key Laboratory of Freshwater Ecology and Biotechnology (No. 2011FBZ13). Disclaimer—There is no conflict of interest. Data availability—Data, associated metadata, and calculation tools are available on request from the authors ([email protected]).
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REFERENCES 1. Dingemans MM, van den Berg M, Westerink RH. 2011. Neurotoxicity of brominated flame retardants: (in)direct effects of parent and hydroxylated polybrominated diphenyl ethers on the (developing) nervous system. Environ Health Perspect 119: 900-907.
2. Daston G, Faustman E, Ginsberg G, Fenner-Crisp P, Olin S, Sonawane B, Bruckner J, Breslin W, McLaughlin TJ. 2004. A framework for assessing risks to children from exposure to environmental agents. Environ Health Perspect 112: 238-256.
3. Staskal DF, Diliberto JJ, Birnbaum LS. 2006. Disposition of BDE 47 in developing mice. Toxicol Sci 90: 309-316.
4. Eriksson P, Jakobsson E, Fredriksson A. 2001. Brominated flame retardants: a novel class of developmental neurotoxicants in our environment? Environ Health Perspect 109: 903-908.
5. Belles M, Alonso V, Linares V, Albina ML, Sirvent JJ, Domingo JL, Sanchez DJ. 2010. Behavioral effects and oxidative status in brain regions of adult rats exposed to BDE-99. Toxicol Lett 194: 1-7.
6. Yu K, He Y, Yeung LW, Lam PK., Wu RS, Zhou B. 2008. DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells in vitro. Toxicol Sci 104: 341-351.
7. Alm H, Scholz B, Fischer C, Kultima K, Viberg H, Eriksson P, Dencker L, Stigson M. 2006. Proteomic evaluation of neonatal exposure to 2,2 ,4,4 ,5-pentabromodiphenyl ether. Environ Health Perspect 114: 254-259.
This article is protected by copyright. All rights reserved
A c c e p t e d P r e p r i nt
8. Rico EP, Rosemberg DB, Seibt K, Capiotti KM, Da Silva RS, Bonan CD. 2011. Zebrafish neurotransmitter systems as potential pharmacological and toxicological targets. Neurotoxicol Teratol 33: 608-617.
9. Dreiem A, Okoniewski RJ, Brosch KO, Miller VM, Seegal RF. 2010. Polychlorinated biphenyls and polybrominated diphenyl ethers alter striatal dopamine neurochemistry in synaptosomes from developing rats in an additive manner. Toxicol Sci 118: 150-159.
10. Viberg H, Fredriksson A, Eriksson P. 2003. Neonatal exposure to polybrominated diphenyl ether (PBDE 153) disrupts spontaneous behaviour, impairs learning and memory, and decreases hippocampal cholinergic receptors in adult mice. Toxicol Appl Pharmacol 192: 95-106.
11. Dufault C, Poles G, Driscoll LL. 2005. Brief postnatal PBDE exposure alters learning and the cholinergic modulation of attention in rats. Toxicol Sci 88: 172-180.
12. Bradner JM, Suragh TA, Wilson WW, Lazo CR, Stout KA, Kim HM, Wang MZ, Walker DI, Pennell KD, Richardson JR, Miller GW, Caudle WM. 2013. Exposure to the polybrominated diphenyl ether mixture DE-71 damages the nigrostriatal dopamine system: role of dopamine handling in neurotoxicity. Exp Neurol 241: 138-147.
13. Iversen SD, Iversen LL. 2007. Dopamine: 50 years in perspective. Trends Neurosci 30: 188-193. 14. Irons TD, Kelly PE, Hunter DL, MacPhail RC, Padilla S. 2013. Acute administration of dopaminergic drugs has differential effects on locomotion in larval zebrafish. Pharmacol Biochem Behav 103: 792-813.
15. Eddins D, Cerutti D, Williams P, Linney E, Levin ED. 2010. Zebrafish provide a sensitive model of persisting neurobehavioral effects of developmental chlorpyrifos exposure: comparison with
This article is protected by copyright. All rights reserved
A c c e p t e d P r e p r i nt
nicotine and pilocarpine effects and relationship to dopamine deficits. Neurotoxicol Teratol 32: 99-108.
16. Yu L, Deng J, Shi X, Liu C, Yu K, Zhou B. 2010. Exposure to DE-71 alters thyroid hormone levels and gene transcription in the hypothalamic-pituitary-thyroid axis of zebrafish larvae. Aquat Toxicol 97: 226-233.
17. Sallinen V, Torkko V, Sundvik M, Reenila I, Khrustalyov D, Kaslin J, Panula P. 2009. MPTP and MPP+ target specific aminergic cell populations in larval zebrafish. J Neurochem 108: 719-731.
18. Chen Q, Yu LQ, Yang LH, Zhou BS. 2012. Bioconcentration and metabolism of decabromodiphenyl ether (BDE-209) result in thyroid endocrine disruption in zebrafish larvae. Aquat Toxicol 110: 141-148.
19. Yamamoto K, Ruuskanen JO, Wullimann MF, Vernier P. 2010. Two tyrosine hydroxylase genes in vertebrates New dopaminergic territories revealed in the zebrafish brain. Mol Cell Neurosci 43: 394-402.
20. Suen MFK, Chan WS, Hung KWY, Chen YF, Mo Z.X, Yung KKL. 2013. Assessments of the effects of nicotine and ketamine using tyrosine hydroxylase-green fluorescent protein transgenic zebrafish as biosensors. Biosens Bioelectron 42: 177-185.
21. Chen YC, Priyadarshini M, Panula P. 2009. Complementary developmental expression of the two tyrosine hydroxylase transcripts in zebrafish. Histochem Cell Biol 132: 375-381.
22. Tay TL, Ronneberger O, Ryu S, Nitschke R, Driever W. 2011. Comprehensive catecholaminergic projectome analysis reveals single-neuron integration of zebrafish ascending and descending dopaminergic systems. Nat Commun 2.
This article is protected by copyright. All rights reserved
A c c e p t e d P r e p r i nt
23. Elsalini OA, Rohr KB. 2003. Phenylthiourea disrupts thyroid function in developing zebrafish. Dev Genes Evol 212: 593-598.
24. Parng C, Roy NM, Ton C, Lin Y, McGrath P. 2007. Neurotoxicity assessment using zebrafish. J. Pharmacol. Toxicol. Methods 55: 103-112.
25. He J, Yang D, Wang C, Liu W, Liao J, Xu T, Bai C, Chen J, Lin K, Huang C, Dong Q. 2011. Chronic zebrafish low dose decabrominated diphenyl ether (BDE-209) exposure affected parental gonad development and locomotion in F1 offspring. Ecotoxicology 20: 1813-1822.
26. Luo Q, Cai ZW, Wong MH. 2007. Polybrominated diphenyl ethers in fish and sediment from river polluted by electronic waste. Sci Total Environ 383: 115-127.
27. Chen L, Huang C, Hu C, Yu K, Yang L, Zhou B. 2012. Acute exposure to DE-71: Effects on locomotor behavior and developmental neurotoxicity in zebrafish larvae. Environ. Toxicol Chem 31: 2338-2344.
28. Stapleton HM, Letcher RJ, Baker JE. 2004. Debromination of polybrominated diphenyl ether congeners BDE 99 and BDE 183 in the intestinal tract of the common carp (Cyprinus carpio). Environ Sci Technol 38: 1054-1061.
29. Mariussen E, Fonnum F. 2001. The effect of polychlorinated biphenyls on the high affinity uptake of the neurotransmitters, dopamine, serotonin, glutamate and GABA, into rat brain synaptosomes. Toxicology 159: 11-21.
30. Rink E, Wullimann MF. 2002. Development of the catecholaminergic system in the early zebrafish brain: an immunohistochemical study. Dev Brain Res 137: 89-100.
31. Jaber M, Jones S, Giros B, Caron MG. 1997. The dopamine transporter: A crucial component This article is protected by copyright. All rights reserved
A c c e p t e d P r e p r i nt
regulating dopamine transmission. Movement Disord 12: 629-633.
32. Caudle WM, Richardson JR, Delea KC, Guillot TS, Wang MZ, Pennell KD, Miller GW. 2006. Polychlorinated biphenyl-induced reduction of dopamine transporter expression as a precursor to Parkinson's disease-associated dopamine toxicity. Toxicol Sci 92: 490-499.
33. Buske C, Gerlai R. 2011. Early embryonic ethanol exposure impairs shoaling and the dopaminergic and serotoninergic systems in adult zebrafish. Neurotoxicol Teratol 33: 698-707.
34. Chen Q, Huang N, Huang J, Chen S, Fan J, Li C, Xie F. 2009. Sodium benzoate exposure downregulates the expression of tyrosine hydroxylase and dopamine transporter in dopaminergic neurons in developing zebrafish. Birth Defects Res Part B, Dev Reprod Toxicol 86: 85-91.
35. Airavaara M, Shen H, Kuo CC, Peranen J, Saarma M, Hoffer B, Wang Y. 2009. Mesencephalic Astrocyte-Derived Neurotrophic Factor Reduces Ischemic Brain Injury and Promotes Behavioral Recovery in Rats. J Comp Neurol 515: 116-124.
36. Chen Y, Sundvik M, Rozov S, Priyadarshini M, Panula P. 2012. MANF regulates dopaminergic neuron development in larval zebrafish. Dev Biol 370: 237-249.
37. De Felice E, Porreca I, Alleva E, De Girolamo P, Ambrosino C, Ciriaco E, Germana A, Sordino P. 2014. Localization of BDNF expression in the developing brain of zebrafish. J Anat 224: 564-574.
38. Diekmann H, Anichtchik O, Fleming A, Futter M, Goldsmith P, Roach A, Rubinsztein DC. 2009. Decreased BDNF levels are a major contributor to the embryonic phenotype of huntingtin knockdown zebrafish. J Neurosci 29: 1343-1349.
This article is protected by copyright. All rights reserved
A c c e p t e d P r e p r i nt
39. Luo G, Chen Y, Li X, Liu T, Le W. 2008. Nr4a2 is essential for the differentiation of dopaminergic neurons during zebrafish embryogenesis. Mol Cell Neurosci 39: 202-210.
40. Usenko CY, Robinson EM, Usenko S, Brooks BW, Bruce ED. 2011. PBDE developmental effects on embryonic zebrafish. Environ Toxicol Chem 30: 1865-1872.
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A c c e p t e d P r e p r i nt
Figure 1. PBDEs accumulate in zebrafish larvae exposed to DE-71. The concentration of different PBDE congeners were measured in zebrafish larvae exposed to 0, 3, 10, 30, and 100 g/L DE-71 for 5 days. n = 3 replicate samples; each sample contained 100 larvae; data expressed as mean ± SEM. Figure 2. DE-71 exposure is associated with a reduced whole-body content of dopamine and dihydroxyphenylacetic
acid.
The
concentration
of
(A)
dopamine
(DA)
and
(B)
dihydroxyphenylacetic acid (DOPAC) were measured in zebrafish larvae exposed to 0, 3, 10, 30, and 100 g/L DE-71 for 5 days. n = 3 replicate samples; each sample contained 50 larvae; data expressed as mean ± SEM; *P < 0.05, and ** P < 0.01 indicate significant differences between the exposed larvae and the control group. Figure 3. DE-71 exposure is associated with reduced protein expression levels of tyrosine hydroxylase and the dopamine transporter. Western blot analysis was used to measure the protein expression level of tyrosine hydroxylase (TH; A and B) and the dopamine transporter (DAT; C and D) in zebrafish larvae exposed to 0, 3, 10, 30, and 100 g/L of DE-71 for 5 days. Upper panel shows a representative western blot and the lower panel shows quantification of three replicate samples, normalized to GAPDH expression; data expressed as mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001 indicate significant differences from the control group. Figure 4. Decreased tyrosine hydroxylase expression in the forebrain of zebrafish larvae exposed to DE-71. Whole-mount immunofluorescence was used to assess tyrosine hydroxylase (TH) expression of dopaminergic neurons in zebrafish larvae after exposure to 0, 3, 10, 30, and 100 g/L of DE-71 for 5 days. (A) The control larvae; (B) 100 g/L DE-71 exposure larvae; (C) TH expression levels are indicated by fold change of fluorescence intensity relative to the control. The abbreviations are used: olfactory bulb (Ob), telencephalon (Tel), ventral diencephalon (vDc) and caudal hypothalamus (Hc). Data are presented as mean ± SEM from 6 individual larvae in each group; ** P < 0.01; *** P < 0.001 significant difference from the control group. Figure 5. Altered expression of neurodevelopmental genes in zebrafish larvae exposed to DE-71. The mRNA expression level of manf, bdnf and nr4a2b were significantly decreased following exposure 0,
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3, 10, 30, and 100 g/L DE-71 for 5 days in a concentration dependent manner. Data presented as mean ± SEM; n = 3 replicate samples; each sample contained 30 larvae; ** P < 0.01, *** P < 0.001 indicate significant differences between the exposed larvae and the control group. Figure 6. Abnormal locomotor behavior in zebrafish larvae exposed to DE-71. Locomotor behavior was assessed in zebrafish larvae exposed to 0, 3, 10, 30, or 100 µg/L DE-71 for 5 days. The following parameters were measured: average swimming speed during a continuous light test (A); locomotor traces of the larvae (B); and average swimming speed of the larvae during a light-dark-light-dark photoperiod stimulation test (C). Data are expressed as the mean ± SEM of three replicates (ten larvae per replicate) in 30-s intervals; * P < 0.05, *** P < 0.001 indicate significant difference between the exposed larvae and the control group.
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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